This invention relates to promoter probe vectors, which can be used for readily determining the presence of efficient promoter regions on DNA segments, which regions are effective in selected species of microorganisms to permit microbiological expression of genetic information. More particularly, the invention relates to promoter probe vectors, which are capable of providing easy identification of efficient promoters for expression of genes in Bacillus subtilis and Escherichia coli, as well as methods of making and using such vectors, and products made utilizing such vectors.
A primary basis of industrial utility of recombinant DNA inventions and developments is production of desired products by microorganisms which are normally unable to produce them. In this regard, much of the published literature concerning the application of genetic engineering techniques for the production of commercially valuble products, such as hormones, vaccines, enzymes and various polypeptides, involves use of Escherichia coli as the host, into which foreign genes are introduced. Nevertheless, many fermentation engineers consider Bacillus subtilis to be the bacterium of choice for industrial application of recombinant DNA technology because of the likelihood of increased yield and decreased toxicity of products made by recombinant B. subtilis organisms.
In Escherichia coli and other organisms, the use of operon fusions has proved to be a powerful tool for understanding gene regulation. Operon fusion is a technique where a gene coding for a desired product is fused to the regulatory region of a different gene operon, thereby using that operon's promoter region to cause expression of the desired product. Expression of a foreign gene in a given host often requires that that gene be transcribed from a promoter site indigenous to the host, a factor which has important ramifications for many practical applications of recombinant DNA technology.
At the present state of this technology, it is difficult if not impossible to predict which promoter segments will be operable in a given type of bacterium for expression of a given gene. Yet commercial scale use of the genetic information obtained through recombinant DNA techniques applied to Escherichia coli or other microorganisms may require means for obtaining efficient expression of a desired gene in a microorganism like Bacillus subtilis.
Until the present invention, location of promoters which permit efficient expression of target genes in B. subtilis has been a tedious, difficult procedure, with uncertain results. Those plasmids which have been utilized to achieve expression of foreign genes in B. subtilis have typically relied on expression of antibiotic resistance properties, and the techniques utilized to judge the efficiency of expression have involved growth measurement of the rDNA recipient microorganisms in the presence of the antibiotic for which resistance would indicate that expression had been achieved. (e.g. chloramphenicol, tetracycline, penicillin, kanamycin). Other techniques, such as spectrophotometric analysis for chloramphenicol acetyl transferase (CAT) are also complex and time consuming, require lysis or destruction of the bacteria being assayed, and are difficult to apply to the usual multitude of assay samples. See Goldfarb, et al, "Expression of Tn9-derived chloramphenicol resistance in Bacillus subtilis," Nature 293:309, 310 (Sept. 24, 1981); Williams, et al., "Cloning Restriction Fragments That Promote Expression of a gene in Bacillus subtilis," J. Bact. 146:1162 (June, 1981). The plasmids and methods of the present invention, however, provide means for identifying efficient promoters in DNA segments in a simple, rapid manner, with essentially immediately determinable results.